Proteins and peptides are naturally occurring heteropolymers composed of amino acids. Proteins are found in all living cells and function as structural and transport elements, catalytic enzymes and hormones. The order of the amino acids in the protein chain (primary structure) ultimately determines the complex three dimensional solution structure adopted by a protein that is necessary for active biological function. Research directed towards the structure and function of particular proteins often requires that the primary structure (sequence) be determined.
Recently there has been considerable interest in sequencing proteins which are available in much smaller amounts. These proteins include growth factors, peptide hormones, cell membrane receptors and proteins involved in intracellular signal transduction. The complete primary structures of these low-abundance proteins is now frequently determined using combinations of protein chemical and recombinant DNA methods. High sensitivity protein sequencing is also employed in the identification and isolation of the coding gene from appropriate gene libraries (Ullrich, A. et al., Nature 313:756 (1985).
For more than thirty years the order of amino acids in a protein chain has been determined by stepwise chemical or enzymic processes in which single amino acids are selectively removed one by one from either the amino- or carboxy- terminal end. The preferred chemical degradation method was introduced by Edman (Acta Chem. Scand. 4:283 (1950), but other methods have been developed and can be usefully employed in other instances.
In the Edman process, the protein is first reacted at its amino terminus with an isothiocyanate (e.g., phenylisothiocyanate, PITC) under basic conditions to form the phenylthiourea. Treatment with anhydrous acid (e.g., trifluoracetic acid, TFA) causes cyclization of the phenylthiourea to the anilinothiazolinone with concomitant cleavage of the N-terminal amino acid fromthe polypeptide and exposure of the alpha amine of the next, adjacent amino acid in the chain. The free ATZ amino acid is extracted, converted to the more stable phenylthiohydantoin (PTH) derivative by treatment with aqueous acid (e.g., 20% v/v aqueous TFA) and identified by chromatographic means (e.g., reverse-phase high pressure liquid chromatography (HPLC) or thin-layer chromatography). The parent polypeptide, now shortened by one residue, is subjected to repeat reaction with PITC and TFA to furnish the next ATZ amino acid in sequence.
The repetitive nature of the process lends itself readily to automation, and several automated sequencing machines have been developed over the past 25 years, all implementing variations of the basic Edman degradation chemistry. Several of these machines are the liquid-phase sequencer developed by Edman and Begg (Edman. P., and Begg, G., Eur. J. Biochem. 1:20 (1967) and U.S. Pat. No. 3,725.010), the solid-phase sequencer (Laursen R., Eur. J. Biochem. 20:89 (1971) and the gas-phase sequencer (Hewick, R. M. et al., J. Biol. Chem. 256:7990 (1981)).
The solid-phase process described by Laursen is distinct from the gas- and liquid-phase implementations of the Edman chemistry in that the polypeptide sample is covalently immobilized to a solid support before being subjected to the degradation chemistry. Attractive advantages to this latter approach stem from the fact that proteins which are covalently linked to an insoluble matrix can readily be separated, without extractive losses, from reagent and reaction by-products. This leads directly to shorter instrument cycle times, higher stepwise sequencing efficiencies and significant reduction in the UV-absorbing background contaminants that can interfere with the identification of PTH amino acids by reverse-phase HPLC. The solid-phase approach also allows for considerable flexibility in the choice of reagents, solvents and reaction conditions necessary for the development of highly efficient sequencing chemistries (for review see Laursen, R. A. and Machleidt, W., in Methods of Biochemical Analysis 26:201 (1980)).
Numerous derivatized supports have been described for use in solid-phase sequence analysis, including polystyrene beads derivatized with both aryl and alkyl- primary amines (Laursen, R. A. Eur. J. Biochem. 20:89 (1971)), controlled pore glass beads derivatized with alkyl amines, aryl amines and diisothiocyanates (Wachter, E., et al., FEBS Lett. 35:97 (1973); Weetall, H. H., Biochem. Biophys. Acta 212:1 (1970)) and both glass fiber sheets and polyvinylidene difluoride membranes derivatized with diisothiocyanates (Aebersold, R., et al., J. Biol. Chem. 261:4229 (1986); Coull, J., et al., in Methods in Protein Sequence Analysis (1989) Wittman-Liebold, B., (Ed.) Springer-Verlag, Berlin). Proteins and peptides have been successfully covalently attached to all the above derivatized supports and analysed by solid-phase Edman degradation.
One feature of these classical approaches to achieving covalent attachment of proteins and peptides to an insoluble matrix was that the matrix surface itself was derivatized with chemical groups involved in the linking process. In a more unconventional approach, Tarr described a process whereby proteins or peptides could be covalently immobilized on the interior surfaces of glass capillaries by the formation of a polymer network comprised of protein and polyamine polymers covalently crosslinked with 1,4-phenylenediisothiocyanate (Tarr, G. E., J. Protein Chem. 7:293 (1988)). Tarr noted that with glass capillaries the resulting polymer network was effectively immobilized on the walls of the capillaries even in the absence of covalent linkages between the polymer network and the glass. This was not the case for plastic capillary surfaces, where the polymer network had to be covalently linked to the surface to prevent desorption from the tubing walls during the sequencing chemistry.
Incremental improvements in instrumentation and laboratory techniques over the past two decades (see Wittman-Liebold, B., (Ed.) in Methods in Protein Sequence Analysis (1989) Springer Verlag, Berlin) have resulted in automated sequencing machines which can routinely provide useful sequence information at the level of only a few picomoles of protein (Kent, S., et al., BioTechniques 5:314 (1987)).
The high-yield purification of such small amounts of protein in a form suitable for sequence analysis presents a considerable technical challenge to the protein chemist. The more traditional purification techniques involving gel permeation, thin-layer and ion-exchange chromatography have increasingly given way to narrow and microbore implementations of high-pressure liquid chromatography (e.g., size exclusion, ion-exchange and reverse-phase) (see Wilson, K. J. and Yuan, P. M. in Protein Sequencing: A Practical Approach (1989) Findlay, J. B. C. and Geisow, M., (Eds.) IRL Press, Oxford and New York). The extremely high resolving power of one- and two-dimensional polyacrylamide gel electrophoresis has also attracted recent attention, particularly when combined with the transfer of separated proteins from the acrylamide or agarose gel matrix to sheets of nitrocellulose, nylon or hydrophobic polymers (`Western` blotting).
Early efforts to recover proteins from the separating gel matrices by direct electroelution from gel slices often resulted in low sample recovery, protein degradation and amino-terminal blocking (Bhown, A. S., et. al., Analyt. Biochem. 103:184 (1980); Hunkapillar, M. W. and Lujan, E., in Methods of Protein Microcharacterization, (1986) Shively, J. E., (Eds.), Humana Press, Clifton, NJ). Protein samples recovered from gel slices in this fashion were often also contaminated with large quantities of SDS detergent and buffer salts, the removal of which was often tedious and associated with significant loss of material.
Conventional diffusion blotting from polyacrylamide gels to nitrocellulose or nylon sheets was easy to perform, but protein recoveries were often low, particularly for larger proteins coated with SDS (Lee, C. Y., et al., Analt. Biochem. 123:14 (1982)).
Following the initial study by Towbin, H. et al, (Proc. Natl. Acad. Sci. USA 76:4350 (1979), electrophoretic transfer of proteins from gels to a sheet matrix has become the most widely used technique for protein blotting as it offers highly efficient transfer of even high molecular weight proteins within a short period of time. Once electrophoretically transferred to the blotting support the non-covalently adsorbed proteins could be visualized by staining with conventional dyes such as Coomassie brilliant Blue, Ponceau S, India ink or Amido Black. Contaminating buffer salts and detergents could also be rinsed from the surface without removal of the bound proteins, particularly when using hydrophobic polymer membranes such as polyvinylidene difluoride (PVDF) as the blotting matrix (Pluskal, M., et al., BioTechniques 4:272 (1986)).
The traditional blotting membranes such as nitrocellulose or nylon are destroyed by exposure to organic solvents and are therefore unsuitable for use in the direct sequence analysis of electroblotted proteins. Aebersold, R., et al., (J. Biol. Chem. 261:4229 (1986)) pioneered the use of chemically modified glass-fiber sheets as blotting supports that could be used for direct sequence analysis in gas-phase sequencers (see also Vanderkerkhove, J., et al., Eur. J. Biochem. 152:9 (1985)). These supports, derivatized with primary amines, quaternary amines or quaternized ammonium polybases added significant protein binding capacity to the base glass-fiber sheets but proved difficult to prepare and gave great variance in performance between laboratories. Severe problems were also encountered in visualizing the transferred proteins as the chemically modified glass-fiber surfaces readily bound the common protein staining agents described above and hence prevent the proteins from being detected.
The use of PVDF membranes for electroblotting and direct sequence analysis by P. Matsudaira (J. Biol. Chem. 261:10035 (1987) overcame many of the problems associated with the coated glass-fiber supports. This membrane has now emerged as the preferred substrate for electroblotting/sequencing applications and improvements to the original method have already been reported (Xu, Q. and Shively. J. E., Analyt. Biochem. 170:19 (1988) Speicher, D. W. in Techniques in Protein Chemistry, (1989) Hugli, T., (Eds.), Academic Press, San Diego).
The base membrane material (polyvinylidene difluoride) is a teflon-like polymer which is both mechanically strong and chemically inert. Protein transfer to the membrane is straightforward. In addition, the surface of the membrane demonstrates high protein binding capacity (up to 170 .mu.g/cm.sup.2). A key advantage is the ability to stain and destain the membrane using general protein dyes. Sequence data has been obtained on proteins detected with Coomassie Blue, Amido Black and reverse staining with Ponceau S (LeGendre, N. and Matsudaira, P., BioTechniques 6:154 (1988)). A major restriction that has prevented more widespread acceptance of solid-phase sequencing methods has been that the beaded supports (polystyrene or controlled pore glass, described above) or the capillary immobilization method described by Tarr for solid-phase sequencing are not compatible with the more direct electroblotting methods now commonly used for the preparation of samples for sequence analysis.
Proteins bound to the surfaces of the primary or quaternary amine-modified glass fiber sheets or PVDF membranes are immobilized by non-covalent interactions between protein and surface. The interaction is primarily by hydrophobic and/or weak coulombic attraction. However, protein sequence analysis of samples on these supports could only be performed using adsorptive or gas-phase sequencing chemistries that did not require a covalent linkage between the protein and surface. In an effort to utilize some of the favorable characteristics of solid-phase sequence analysis described previously, Aebersold, R. et al. (J. Biol. Chem. 261:4229 (1986)) covalently immobilized proteins by electroblotting onto glass-fiber sheets derivatized with phenylenediisothiocyanate (DITC). Significant drawbacks to this use of the glass fiber paper include the non-optimal surface texture that does not allow for intimate contact with the gel and undistorted transfer of protein during the blotting process, the low protein binding capacity (7-10 ug/cm.sup.2), and the fact that both the initial and repetitive sequencing yields for proteins electroblotted onto derivatized glass-fiber sheets decreases with a decreasing amount of protein applied (Yuen. S , et al., Applied Biosystems User Bulletin 24 (1986). This approach was significantly improved by Coull, J., et al., (Methods in Protein Sequence Analysis (1989), Wittman-Liebold, B., (Ed.), Springer-Verlag, Berlin; U.S. patent application Ser. No. 07/212,430, filed June 28, 1988. entitled "Membranes for Solid Phase Protein Sequencing") using PVDF membranes modified with diisothiocyanates and now a U.S. Pat. No. 5,011,861. Initial expectations that these approaches might lead both to increased blotting and sequencing efficiencies were only partially realized. The major disadvantages were the high cost of preparing diisothiocyanate derivatized membrane surfaces in sufficient quantity for electroblotting applications and the significant problems associated with staining the electroblotted proteins on the chemically modified surfaces.